Methods and compositions for attenuating antibody-mediated xenograft rejection in human recipients

ABSTRACT

Antibody-mediated xenograft rejection is attenuated by (1) removing preformed antibodies to various identified carbohydrate xenoantigens from the recipient&#39;s circulation prior to transplantation by extracorporeal perfusion of the recipient&#39;s blood over a biocompatible solid support to which the xenoantigens are bound and/or (2) parenterally administering a xenoantibody-inhibiting amount of an identified xenoantigen to the recipient shortly before graft revascularization and thereafter.

This application is a divisional of application Ser. No. 07/933,466,filed Aug. 21, 1992 which, in turn, is a continuation-in-part of U.S.application Ser. No. 07/749,529 filed Aug. 23, 1991, now abandoned,which application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention is in the general field of transplantationimmunology and relates specifically to xenotransplantation and tocompositions and methods for facilitating xenotransplantation in humansthrough inhibition and/or removal of preformed human antibodies tocarbohydrate xenoantigens.

REFERENCES

The following references are cited in the application as superscriptnumbers at the relevant portion of the application.

1. Agashi, T.: Presentation at American Society for Artificial InternalOrgans, 37th Annual Meeting in Chicago: Apr. 26, 1991.

2. Bannett, A. D., McAlack, R. P., Raja, R., Baquero, A., Morris, M.:Transplant. Proc. XIX:4543-4546, 1987.

3. Bensinger, W. I., Buckner, C. D., Thomas, E. D., Clift, R. A.:Transplantation. 33:427-429, 1982.

4. Chien, J. L., Li, S. C., Li, Y. T.: J. Lipid Res. 20:669-673, 1979.

5. Dubois, M., Gilles, K., Hamilton J. K., Rebers, P. A., Smith, F.:Anal. Chem. 28:350-356, 1956.

6. Egge, H., Kordowicz, M., Peter-Katalinic, J., Hanfland, P.: J. Biol.Chem. 260:4927-4935, 1985.

7. Eto, T., Ichikawa, Y., Nishimura, K., Ando, S., Yamakawa, T.: J.Biochem. (Tokyo) 64:205-213, 1968.

8. Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., Macher, B. A.:Proc. Natl. Acad. Sci. 84: 1369-1373, 1987.

9. Galili, U., Macher, B. A., Buehler, J., Shohet, S. B.: J. Exp. Med.162:573-582, 1985.

10. Galili, U., Shohet, S. B., Kobrin, E., Stults, C. L., Macher, B. A.:J. Biol. Chem. 263:17755-17762, 1988.

11. Holgersson, J., Cairns, T. D. H., Breimer, M. E., Taube, D., Welsh,K., Samuelsson, B. E.: Glycoconjugate J. 8:172, 1991.

12. Holgersson, J., Jovall, P. A., Samuelsson, B. E., Breamer, M. E.: J.Biochem. (Tokyo) 108:766, 1990.

13. Lemieux, R. U., Baker, D., Bundle, D.: U.S. Pat. No. 4,137,401,issued Jan. 30, 1979.

14. Lemieux, R. U., Baker, D., Bundle, D.: U.K. Patent No. 1544908,issued Aug. 29, 1979.

15. Lemieux, R. U., Bundle, D., Baker, D. A.: U.S. Pat. No. 4,238,473,issued Dec. 9, 1980.

16. Lemieux, R. U., Ratcliffe, R. M.: U.S. Pat. No. 4,362,720, issuedDec. 7, 1982.

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20. Platt, J. L., Lindman, B. J., Chen, H., Spitalnik, S. L., Bach, F.H.: Transplant. 50:817-822, 1990.

21. Rapp, H. J., Borsos, T.: J. Immunol. 96:913-919, 1966.

22. Stellner, K., Hakomori, S., Warner, G. A.: Biochem. Biophys. Res.Commun. 55:439-445, 1973.

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24. Weetall, H. H.: Methods in Enzymology XLIV:140, 1976.

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26. Dahmen, J., Torbjorn, F., Magnusson, G., Noori, G., Carlstrom, A.:Carbohydr. Res. 127:15-25, 1984.

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29. Jacquinet, J., Duchet, D., Milat, M., Sinay, P.: J.C.S. PerkinI:326-330, 1981.

30. Koike, K., Sugimoto, M., Sato, S., Ito, Y., Nakahara, Y., Ogawa, T.:Carbohydr. Res. 163:189-208, 1987.

31. Schaubach, R., Hemberger, J., Kinzy, W.: Liebigs Ann. Chem. 607-614,1991.

32. Laus, R., Ulrichs, K., Muller-Ruchholtz, W.: Int. Archs. AllergyAppl. Immun. 85:201-207, 1988.

33. Ratcliffe et al., U.S. Pat. No. 5,079,353, issued Jan. 7, 1992, for"Sialic Acid Glycosides, Antigens, Immunoadsorbents, and Methods forTheir Preparation".

34. Okamoto et al., Tetrahedron, Vol. 46, No. 17, pp. 5835-5857 (1990).

35. Abbas et al., Proc. Japanese-German Symp. Berlin, pp. 20-21 (1988).

36. Paulsen, Angew. Chem. Int. Ed. Eng., 21:155-173 (1982).

37. Schmidt, Angew. Chem. Int. Ed. Eng., 25:212-235 (1986).

38. Fugedi et al., Glycoconj. J., 4:97-108 (1987).

39. Kameyama et al., Carbohydr. Res., 209:C₁ -C₄ (1991).

40. Ekborg et al., Carbohydr. Res. 110:55-67 (1982).

41. Dahmen et al., Carbohydr. Res. 118:292-301 (1983).

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48. Ratcliffe et al., U.S. Pat. No. 5,344,870.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

BACKGROUND

Over 15,000 organ transplants were performed in the U.S.A. in 1990.(Annual Report of the U.S. Scientific Registry for Organ Transplantationand the Organ Procurement and Transplantation Network. 1990.) Theappropriate organs were taken from 6,000 donors, of whom fewer than4,500 were cadaveric donors. The number of patients on the waiting listof the United Network of Organ Sharing in the U.S.A. at any one timeapproximates 23,000. Therefore, many potential recipients will bewaiting for periods considerably in excess of one year for suitableorgan transplants.

As the success of organ transplantation increases steadily, more andmore patients are being referred for these procedures, and the shortageof suitable organs is becoming ever more acute. At the present time,kidney transplantation is associated with a 1-year graft survival ofwell over 90%, heart transplantation with a graft survival of over 80%,and liver and pancreas transplantation with graft survival ratesapproaching 80%.

Despite major efforts at educating the public and the medical professionwith regard to the need for suitable donors, the gap between the demandand the availability of suitable organs is likely to increase. An answerto this problem would be the use of animal organs. Non-human primateshave been considered for use as donors in this context.

However, these animals are in short supply worldwide, and, particularlywith regard to the larger apes, are frequently endangered species. Theyare, therefore, not numerous enough to be considered in this role, and,furthermore, the numbers could not easily be increased even bywidespread captive breeding programs. Other disadvantages includerelatively small size, making them unsuitable as donors of organs foradult humans, and the risk of viral infection. There would also bevociferous public opposition to the use of these animals on asignificant scale.

More distant mammalian species, such as the pig, would be very suitablein many regards. They grow to an appropriate size, breed easily, can bereared in specific pathogen free herds or even gnotobiotic (germ-free)conditions, and are already bred in large numbers specifically for thepurpose of human consumption. Organ transplantation between widelydisparate species, such as pig and man, however, is followed byantibody-mediated hyperacute rejection within minutes or hours, and thisrejection cannot be inhibited or treated by the currently availableimmunosuppressive regimens. If the problem of antibody-mediatedrejection could be overcome, then organ transplantation may no longer berestricted by the number of human donors that become available eachyear.

The benefits to society would be considerable. At present, one-third ofthose awaiting heart transplantation die before a suitable organ becomesavailable. If animal organs were used, patients would be able to undergotransplantation as soon as it was deemed necessary, and the operationscould be performed electively under ideal conditions without the needfor emergency procedures. In addition, many patients are today notaccepted onto transplant waiting lists if they have borderlinecontraindications, as it is felt that the relatively few donor organsthat become available must be used in ideal patients. If there were nolimitation on the number of donor organs, then organ transplantationwould certainly be offered to very many more candidates. Thus,compositions and methods which would facilitate xenotransplantationwould be extremely useful.

There has been some success in facilitating non-xenotransplants betweenABO-mismatched individuals. In human transplantation the extracorporealremoval of naturally occurring anti-A and/or anti-B antibodies using amethod similar to those described in several patents (U.S. Pat. Nos.4,137,401¹³ and 4,238,473¹⁵ ; U.K. Patent 1544908¹⁴ ; U.S. Pat. No.5,149,425¹⁸ ; European Patent Application No. 89311540.2¹⁷) has enabledsuccessful transplantation of kidneys and bone marrow betweenABO-mismatched individuals (Bannett et al. 1987², Bensinger et al.1982³).

Anti-A, anti-B and other anti-carbohydrate antibodies have been involvedin allogeneic transfusion reactions and acute rejection of skin grafts,and transplanted organs. It has therefore been hypothesized thatantibodies to carbohydrate determinants may play a significant role inthe acute rejection of xenografts. Some studies indicate that certaincarbohydrate structures are targets for xenoantibodies (Laus et al.1988³², Platt et al. 1990²⁰, Holgersson et al. 1991¹¹).

Numerous glycolipids have been purified from mammalian cells and many ofthese structures are reviewed in a paper by Stults and associates(1989)²³. Linear B type 2-like glycosphingolipids have been purifiedfrom cells obtained from rabbit, cattle, and new world monkeys (Eto etal. 1968⁷, Stellner et al. 1973²², Chien et al. 1979⁴, Egge et al. 1985⁶and Galili et al. 1987⁸).

Numerous specificities of anti-carbohydrate antibodies have beenidentified in plasma from humans. Galili and associates (1985)⁹ haveidentified that anti-αGal(1-3)βGal antibodies constitute as much as 1%of circulating human IgG. This group purified antibodies from human ABsera using αGal(1-3)βGal(1-4)βGlcNAc (linear B type 2) bound tobiocompatible solid supports. They found that these antibodies bound topig endothelial cells (from the aorta), pig epithelial cells (from thelens of the eye) and many other tissues from non-primate mammals and newworld monkeys, but not to tissues from healthy old world monkeys, apesor man (Galili et al. 1988¹⁰).

In non-xenogeneic transplants, the neutralization or removal ofanticarbohydrate antibodies utilizing A and B blood group trisaccharidescovalently attached to a solid support in the form of an immunoadsorbentfor the extracorporeal depletion of human anti-A and anti-B antibodieshas been shown to facilitate kidney and bone marrow transplantationacross the ABO blood group barrier (Bannett et al. 1987², Bensinger etal. 1982³ and Agashi 1991¹). This approach is currently in clinicaltrials. An injectable form of the A and B blood group trisaccharides forthe in situ neutralization of anti-A and anti-B antibodies is currentlyin preclinical development. In studies of xenospecific antibodyactivity, utilization of other oligosaccharides covalently attached to asolid support was previously reported as not being particularlysuccessful for removal of anticarbohydrate antibodies (Laus et al.³²)

DISCLOSURE OF THE INVENTION

The invention encompasses two techniques for facilitatingtransplantation of xenogeneic cells, tissues, or organs into humans. Onetechnique involves extracorporeal removal of xenoantibodies from therecipient's blood. The other involves inhibiting xenoantibodies in vivo.The invention also encompasses compositions that are used in or resultfrom these methods.

Accordingly, one aspect of the invention is a method for attenuatingantibody-mediated xenograft rejection in a human recipient of axenograft comprising: identifying one or more xenoantigens, attachingthe xenoantigen(s) to a biocompatible solid support, withdrawingantibody-containing body fluid from the recipient, removing preformedantibodies to at least one carbohydrate antigen of said xenograft thatis involved in the rejection from the withdrawn body fluid byextracorporeal perfusion of the body fluid over a biocompatible solidsupport to which the antigen(s) is bound through a compatible linkerarm, and reintroducing the perfused body fluid into the recipient.

Another aspect of the invention is a method for attenuatingantibody-mediated xenograft rejection in a human recipient of axenograft comprising identifying at least one carbohydrate xenoantigento preformed antibodies, and parenterally administering at least onecarbohydrate xenoantigen capable of binding one or more antibodies thatis involved in the rejection to the recipient in an mount sufficient toinhibit the recipient's antibodies to the antigen.

A further aspect of the invention is a composition useful forattenuating antibody-mediated xenograft rejection in a human recipientof a xenograft comprising an injectable formulation of at least onecarbohydrate xenoantigen.

Yet another aspect of the invention is an immunoadsorbent compositionuseful for removing xenoantibodies from the blood of a human recipientof a xenograft to attenuate the rejection of the xenograft by therecipient comprising a biocompatible solid support having at least oneidentified xenoantigen attached thereto through a compatible linker arm.

Still another aspect of the invention is human blood or plasma usefulfor infusing into a human recipient of a xenograft to attenuaterejection of said xenograft, the blood or plasma being depleted ofpreformed antibodies to at least one identified xenoantigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H illustrate carbohydrate structures used to study the bindingof human antibodies eluted from pig heart, pig kidney and/or pig redcell stroma.

FIG. 2 illustrates that antibody-mediated lysis of pig erythrocytes isreduced by preadsorption of human plasma on a variety of matrix-boundcarbohydrates and mixtures of matrix-bound carbohydrate structures.

FIG. 3 illustrates that antibody-mediated lysis of sheep, pig and bovineerythrocytes is reduced by pre-adsorption of human plasma withmatrix-bound carbohydrate and mixtures of matrix-bound carbohydratestructures.

FIGS. 4A and 4B illustrate a reduction in lysis of a variety of pig celltypes when human plasma {O plasma (A) and AB plasma (B)} werepre-adsorbed with matrix-bound αGal(1-3)βGal(1-4)βGlcNAc-R (linear Btype 2) or αGal(1-3)βGal(1-4),βGlc-R (linear B type 6).

FIG. 5 illustrates a reduction in lysis to a pig kidney cell line(LLC-PK₁) when human plasma (O, A, B and AB) were preadsorbed with somematrix-bound carbohydrate antigens (formulas 2, 4, 6, 9, 13, 18, 31, 33,35, and 36 of FIGS. 1A, 1B, 1C, 1D, 1G and 1H).

FIG. 6 illustrates a reduction in lysis to a pig kidney cell line(LLC-PK₁) when different human AB plasma were preadsorbed with somematrix-bound carbohydrate antigens.

FIG. 7 illustrates a reduction in lysis to a pig kidney cell line(LLC-PK₁) when human plasma (O, A, B and AB) were preadsorbed with somematrix-bound carbohydrate antigens (formulas 2, 3, 19, 20, 34, 37, 38and 40 of FIGS. 1A, 1E, 1G, and 1H).

FIGS. 8A and 8B illustrate a reduction in lysis to a pig kidney cellline (LLC-PK₁) when human plasma {O plasma (A) and A plasma (B)} wereincubated with some soluble carbohydrate antigens.

FIG. 9 illustrates a reduction in lysis to a pig kidney cell line(LLC-PK₁) when human plasma (O, A, B and AB) were incubated with somesoluble carbohydrate antigens.

FIG. 10 illustrates a reduction in lysis to a pig kidney cell line(LLC-PK₁) when different human AB plasma were incubated with somesoluble carbohydrate antigens.

FIGS. 11A-11D illustrate removal of antibodies when human plasma (O, A,B and AB) were preadsorbed with some matrix-bound carbohydrate antigensas detected by ELISA.

FIGS. 12A-12D illustrate removal of antibodies when different human ABplasma were preadsorbed with some matrix-bound carbohydrate antigens asdetected by ELISA.

MODES FOR CARRYING OUT THE INVENTION

A. Definitions

As used herein the following terms have the following meanings:

Xenograft: a cell, tissue, or organ graft of a nonhuman mammalianspecies that normally gives rise to a rejection response in a humanrecipient.

Rejection: an immune response with humoral and/or cellular componentsdirected against a xenograft.

Attenuation: a reduction or elimination of either or both components ofa rejection response.

Antibody-mediated: an immune response that directly or indirectlyresults from antigen-antibody interaction.

Inhibit: a reduction or elimination of the ability of an antibody toinduce a rejection response.

Xenoantigen: a carbohydrate antigen of a xenograft, particularly onethat is involved in a rejection response or an antigen that cross-reactswith an antibody capable of binding to a carbohydrate antigen of axenograft.

Xenoantibodies: pre-formed (existing) human antibodies that recognizeand bind xenoantigens as pan of the rejection response.

Biocompatible: chemical inertness with respect to humanantibody-containing body fluids.

Blood: whole blood, plasma or serum.

Compatible linker arm: a moiety which serves to space the carbohydrateantigen from the biocompatible solid support and which is bifunctionalwherein one functional group is capable of binding to a reciprocalfunctional group of the support and the other functional group iscapable of binding to a reciprocal functional group of the carbohydrateantigen.

B. Extracorporeal Removal of Xenoantibodies from Body Fluids

As indicated, one aspect of this invention involves the steps ofidentifying one or more xenoantigens, attaching the xenoantigen(s) to abiocompatible solid support, withdrawing antibody-containing body fluidfrom a human xenograft recipient, removing preformed antibodies to oneor more xenoantigens from the fluid via affinity chromatography, andreintroducing the xenoantibody-depleted body fluid into the recipient.

While this technique may be applied to any antibody-containing bodyfluid, it will usually be applied to blood. The blood is withdrawn byconventional techniques and an anticoagulant (e.g., heparin, citrate) istypically added to it to prevent coagulation. If desired, cells may beremoved from the blood before it is subjected to xenoantibody depletion.

Xenoantibody depletion is achieved by perfusing the blood over a solidsupport having one or more xenoantigens bound to it. Xenoantigens may beidentified by isolating xenoantibodies from human blood by perfusing theblood over xenograft material, removing bound antibodies from thexenograft, and using those antibodies to screen candidate carbohydrates,such as by a suitable immunoassay. Examples of such xenoantigens are thecarbohydrates listed in Table 1, infra, and shown in FIG. 1A to 1H.

Chemical methods for the synthesis of carbohydrate xenoantigens can beaccomplished by methods known in the art. These materials are generallyassembled using suitably protected individual monosaccharides includingthe desirable glucose, N-acetylglucosamine, galactose,N-acetylgalactosamine, fucose, and rhamnose or lactose intermediate.

The specific methods employed are generally adapted and optimized foreach individual structure to be synthesized. In general, the chemicalsynthesis of all or part of the oligosaccharide glycosides firstinvolves formation of a glycosidic linkage on the anomeric carbon atomof the reducing sugar or monosaccharide. Specifically, an appropriatelyprotected form of a naturally occurring or of a chemically modifiedsaccharide structure (the glycosyl donor) is selectively modified at theanomeric center of the reducing unit so as to introduce a leaving groupcomprising halides, trichloroacetimidate, acetyl, thioglycoside, etc.The donor is then reacted under catalytic conditions well known in theart with an aglycon or an appropriate form of a carbohydrate acceptorwhich possesses one free hydroxyl group at the position where theglycosidic linkage is to be established. A large variety of aglyconmoieties are known in the an and can be attached with the properconfiguration to the anomeric center of the reducing unit.

Appropriate use of compatible blocking groups, well known in the an ofcarbohydrate synthesis, will allow selective modification of thesynthesized structures or the further attachment of additional sugarunits or sugar blocks to the acceptor structures.

After formation of the glycosidic linkage, the saccharide glycoside canbe used to effect coupling of additional saccharide unit(s) orchemically modified at selected positions or, after conventionaldeprotection, used in an enzymatic synthesis. In general, chemicalcoupling of a naturally occurring or chemically modified saccharide unitto the saccharide glycoside is accomplished by employing establishedchemistry well documented in the literature. See, for example, Okamotoet al.³⁴, Ratcliffe et al.³³, Abbas et al.³⁵, Paulson³⁶, Schmidt³⁷,Fugedi et al.³⁸, and Kameyama et al.³⁹, U.S. Pat. Nos. 4,137,401¹³,4,238,473¹⁵, and 4,362,720¹⁶ and in Dahmen et al.²⁶, Schaubach et al.³¹,Garegg et al.²⁷,28, Jacquinet et al.²⁹, Cox et al.²⁵, and Koike et al.³⁰

In the monosaccharide and oligosaccharide glycosides shown in FIGS. 1Ato 1H, X represents oxygen, sulfur, --NH-- or a covalent bond, and Yrepresents hydrogen or an aglycon group (i.e., that component of aglycoside which is not a sugar). In most instances Y represents aradical of the general formula --A--Z wherein A represents a bond, analkylene group of from 2 to 10 carbons, or --(CH₂ --CR₁ --G)_(n) --wherein n is an integer equal to 1 to 5 inclusive; R₁ is selected fromthe group consisting of hydrogen, methyl and ethyl; and G is selectedfrom the group consisting of hydrogen, oxygen, sulfur, nitrogen, phenyl,and phenyl substituted with 1 to 3 substituents selected from the groupconsisting of amine, hydroxyl, halo, and alkyl of from 1 to 4 carbonatoms, and Z is selected from the group consisting of hydrogen, methyland when G is not oxygen, sulfur or nitrogen and A is not a bond, then Zis also selected from the group consisting of OH, SH, --NH₂, NHR₂,--C(O)OR₂, --C(O)NH₂, --C(O)NH--NHR₂, --C(O)NHR₂ and --C(O)N(R₂)₂wherein each R₂ is independently hydrogen or alkyl of from 1 to 4 carbonatoms.

Preferably X represents --O-- and Y represents --A--Z wherein A isalkylene of 2 to 10 carbon atoms and Z is selected from the groupconsisting of --C(O)OR₂, --C(O)NH₂, and --C(O)NHR₂ and R₂ is hydrogen oralkyl of from 1 to 4 carbon atoms.

Particularly preferred carbohydrates from among those shown in FIGS. 1Ato 1H are carbohydrates of formulas 1, 2, 3, 4, 13, 25, 26, 31, and 32wherein X is --O-- and Y is --A--Z where A is alkylene of 2 to 10 carbonatoms and Z is selected from the group consisting of --C(O)OR₂,--C(O)NH₂ and --C(O)NHR₂ where R₂ is hydrogen or alkyl of from 1 to 4carbon atoms.

The solid supports to which the xenoantigens are bound may be in theform of a continuous large surface or in the form of particles. A largevariety of biocompatible solid support materials are known in the art.Examples thereof are silica, synthetic silicates such as porous glass,biogenic silicates such as diatomaceous earth, silicate-containingminerals such as kaolinite, and synthetic polymers such as polystyrene,polypropylene, and polysaccharides. Preferred supports are described inU.S. Pat. No. 5,149,425¹⁸, filed 9 Nov. 1988, the disclosure of which isincorporated herein by reference.

The xenoantigen(s) is covalently bound or noncovalently (passively)adsorbed onto the solid support. The covalent bonding may be viareaction between functional groups on the support and the compatiblelinker arm of the xenoantigen. It has unexpectedly been found thatattachment of the carbohydrate antigen to the biocompatible solidsupport through a compatible linking arm gives effective removal ofanticarbohydrate antibodies. Linking moieties that are used in indirectbonding are preferably organic bifunctional molecules of appropriatelength (at least one carbon atom) which serve simply to distance theantigen from the surface of the solid support. The particular linkingarm is not critical.

Numerous aglycon linking arms are known in the art. For example, alinking arm comprising a para-nitrophenyl group (i.e., --YR=--OC₆ H₄pNO₂) has been disclosed by Ekborg et al.⁴⁰ At the appropriate timeduring synthesis, the nitro group is reduced to an amino group which canbe protected as N-trifluoroacetamido. Prior to coupling to a support,the trifluoroacetamido group is removed thereby unmasking the aminogroup.

A linking arm containing sulfur is disclosed by Dahmen et al.⁴¹Specifically, the linking arm is derived from a 2-bromoethyl groupwhich, in a substitution reaction with thionucleophiles, has been shownto lead to linking arms possessing a variety of terminal functionalgroups such as --OCH₂ CH₂ SCH₂ CO₂ CH₃ and --OCH₂ CH₂ SC₆ H₄ --pNH₂.

Rana et al.⁴² discloses a 6-trifluoroacetamido-hexyl linking arm(--O--(CH₂)₆ --NHCOCF₃) in which the trifluoroacetamido protecting groupcan be removed unmasking the primary amino group used for coupling.

Other exemplification of known linking arms include the7-methoxycarbonyl-3,6,dioxaheptyl linking arm⁴³ (--OCH₂ --CH₂)₂ OCH₂ CO₂CH₃); the 2-(4-methoxycarbonylbutancarboxamido)ethyl⁴⁴ (--OCH₂ CH₂NHC(O)(CH₂)₄ CO₂ CH₃); the allyl linking arm⁴⁵ (--OCH₂ CH═CH₂) which, byradical co-polymerization with an appropriate monomer, leads tocopolymers; other allyl linking arms⁴⁶ are known --O(CH₂ CH₂ O)₂ CH₂CH═CH₂ !. Additionally, allyl linking arms can be derivatized in thepresence of 2-aminoethanethiol⁴⁷ to provide for a linking arm --OCH₂ CH₂CH₂ SCH₂ CH₂ NH₂. Other suitable linking arms are disclosed in U.S. Pat.Nos. 4,137,401¹³, 4,238,473¹⁵, and 4,362,720¹⁶ and in Dahmen et al.²⁶and Garegg et al.²⁷.

Additionally, as shown by Ratcliffe et al.⁴⁸, the R group can be anadditional saccharide or an oligosaccharide containing a linking arm atthe reducing sugar terminus.

Preferably, the aglycon moiety is a hydrophobic group and mostpreferably, the aglycon moiety is a hydrophobic group selected from thegroup consisting of --(CH₂)₈ COOCH₃, --(CH₂)₅ OCH₂ CH═CH₂ and --(CH₂)₈CH₂ OH.

The functional linking arm of the carbohydrate antigen is then used toattach the antigen to a biocompatible solid support. Such attachment iswell known in the art and is disclosed, for example, by Venot et al.,U.S. Ser. No. 07/887,746, (refiled as continuation application Ser. No.08/326,745) which is incorporated herein by reference in its entirety.

Solid supports having combinations of two or more xenoantigens boundthereto may be used to remove xenoantibodies of different specificityfrom the blood or plasma. Alternatively, the blood or plasma may bepassed successively over a series of solid supports each of which hasone or more different xenoantigens bound thereto to removexenoantibodies of different specificity. Accordingly, based on theresults of the examples it may be desirable to use combinations of oneor more linear B type carbohydrate xenoantigens with another type ofcarbohydrate antigen (e.g., an A type antigen, a Forssman type antigen,a Rhamnose antigen, or a glucosaminide type antigen (see FIGS. 1A to1H)). In the case of porcine xenografts, combinations of at least linearB type 2 and linear B type 6 antigens may be preferred. Suchcombinations of antigens may also be employed in the xenoantibodyinhibition procedure described below.

The blood will be contacted with the solid support under conditions thatpromote binding between the xenoantigens bound to the support andcomplementary xenoantibodies present in the blood or plasma. A preferredapparatus and technique for carrying out extracorporeal hemoperfusion isdescribed in said U.S. Pat. No. 5,149,425¹⁸. Contact temperatures in therange of 35° C. to 40° C. are preferably used. The contact time willtypically be in the range of 1 to 6 hr. The unbound portion of the bloodor plasma (i.e., xenoantibody-depleted blood or plasma) is thencollected for reintroduction into the patient or it can be reintroduceddirectly on a continuous basis. The removal of xenoantibodies from therecipient's blood is carried out prior to transplantation (it istypically repeated daily up to 8 times before transplantation) so thatthe xenograft is introduced in the substantial absence of thexenoantibodies or at a time when the xenoantibodies are present atrelatively low titers. The recipient's xenoantibody titer may bemonitored by immunoassay. Insertion of the graft under such conditionslessens the likelihood of antibody-mediated hyperacute rejection (eventhough antibody titers may subsequently increase) and enhances graftsurvival. This phenomenon is variously termed "accommodation,""adaptation," or "anergy." Xenoantibody removal may be continued aftertransplantation if necessary.

Conventional pharmacologic immunosuppression regimes employingnonspecific immunosuppressive agents such as cyclosporine,methylprednisolone, and azathioprine may be employed in conjunction witheither or both of the invention methods.

C. Inhibition of Xenoantibodies In Vivo

As indicated, this technique involves parenterally introducing one ormore identified xenoantigens into the xenograft recipient. Onceintroduced into the circulation, these xenoantigens will bind topre-formed xenoantibodies to neutralize the activity of thosexenoantibodies.

The same xenoantigens that are used in the xenoantibody removaltechnique may be used in the inhibition technique. One or more of thesexenoantigens or pharmaceutically acceptable derivatives thereof (e.g.,esters) is formulated as an injectable in a conventionalpharmaceutically acceptable injectable vehicle (e.g., water forinjection). Formulation typically involves mixing the antigen(s) withthe vehicle, depyrogenating the mix by ultrafiltration, and sterilefiltering the depyrogenated mix. The mix may be lyophilized for storageand reconstituted in sterile vehicle if desired.

The injectable formulation may be administered by intermittent bolusinjection or by continuous intravenous infusion. The administration willtypically be initiated shortly before revascularization of the xenograftand continued for a varying period of time following transplantation.The particular dosing and administration regimen may vary depending uponthe type of transplant, the age, weight, and medical history of therecipient and donor, and the pharmacokinetics of the xenoantigen(s).Typically, the xenoantigens will be administered continuously at dosesranging between about 100 mg/hr and 1000 mg/hr for 1 to 20 days.Concomitant pharmacologic immunosuppression is preferred. Extracorporealremoval of xenoantibodies as described previously may be used incombination with parenteral administration of xenoantigens.

D. Examples

The following examples demonstrate that a wide range of humananti-nonhuman mammalian antibodies are directed at carbohydrate antigenson the cells of a porcine xenograft, that some of these antibodies arecytotoxic (hemolytic) for cells of the xenograft and that application ofmonosaccharides and/or oligosaccharides, either alone or in combination,substantially neutralize these cytolytic antibodies.

In these examples, unless otherwise defined below, the abbreviationsemployed have generally accepted meaning:

ELISA=Enzyme linked immunosorbent assay

BSA=Bovine serum albumin

DMF=dimethylformamide

PBS=Phosphate buffered saline=7.8 mM Na₂ HPO₄, 2.2 mM KH₂ PO₄, 154 mMNaCl and 15 mM NAN₃, pH 7.1 to 7.3

Gal=Galactose

Glc=Glucose

Man=Mannose

GlcNAc=N-acetylglucosamine

GalNAc=N-acetylgalactosamine

Fuc=Fucose

Rha=Rhamnose

FBS=fetal bovine serum

rpm=revolutions per minute

cpm=counts per minute

U=unadsorbed

P=adsorbed with Chromosorb P

LacNAc=βGal(1-4)βGlcNAc-R, R=--(CH₂)₈ --CONH-SYNSORB in Example 3 orR=--(CH₂)₈ --COOCH₃ in Example 4.

SYNSORB=synthetic carbohydrate structures bound to Chromosrb P through a--(CH₂)₈ --CONH-- linking arm.

Reference numbers in FIGS. 4A-12D denote formulas of compounds as setforth in FIGS. 1A to 1H.

These examples are not intended to limit the scope of the invention inany manner.

EXAMPLE 1 Identification of Carbohydrate Compounds Which Bind HumanAnti-mammalian Antibodies

a) Isolation of human "anti-pig" antibodies

Organs from two populations of pigs were used to purify human anti-pigantibodies. The pig strains were Poland China and Yorkshire. Antibodypreparations were obtained by perfusing 100-200 ml of human plasma (O orAB) through pig hearts, pig kidneys or through a column containing solidsupport coated with pig red cell stroma. After washing the tissuesthoroughly with 0.15M NaCl, the retained antibodies were eluted from thetissue or column using 200 ml of 3M NaSCN. The antibodies wereconcentrated, and then the protein content determined.

b) Carbohydrate antigens

Carbohydrate antigens were made by covalent binding of the syntheticoligosaccharides with an aliphatic linking arm (CH₂)₈ COOCH₃ to BSA asdescribed by Pinto and Bundle (1983)¹⁹ and/or Lemieux, Bundle, Baker(U.S. Pat. No. 4,137,401)¹³ (ten to twenty haptens per molecule of BSA).Example 5, infra, describes this procedure as applied to carbohydrate 2of FIG. 1A. Structures and trivial names of some of the monosaccharidesand oligosaccharides used are included in FIGS. 1A to 1H. The use ofsynthetic carbohydrate structures to detect antibody binding has beenpreviously described in U.S. Pat. No. 4,137,401¹³.

c) Enzyme linked immunosorbent assay (ELISA)

The technology used for the ELISA assay for detecting anti-carbohydrateantibodies is a combination of the work from numerous groups. Wells in96 microtitration plates (Flow Laboratories, Inc., McLean Va., U.S.A.)were coated by incubation at room temperature for 16 hours with 2 μg ofBSA-glycoconjugates or BSA per well (20 μg BSA-conjugate/ml diluted inPBS). The coating solutions were removed and 200 μl/well of PBScontaining 5% BSA was added. After a 4 hours incubation (22° C.), thewells were washed twice with PBS, then once with H₂ O. The plates wereinverted, allowed to dry, and stored at room temperature.

Immediately before testing, 200 μl of PBS containing 1% BSA (1% BSA/PBS)was added to each of the wells and left to stand for 10 to 20 minutes atambient temperature. The human anti-pig preparations were diluted tobetween 15 to 70 μg of eluted protein per ml of 1% BSA/PBS. The BSA/PBSwas removed and 100 μl of diluted anti-pig preparation was added to theappropriate wells, then incubated at 4° C. for 16 hours. The antibodypreparations were removed and the wells were washed 4 times with PBS(200 μl/wash). Alkaline phosphatase-conjugated reagents (anti-humanpolyvalent (α, γ and μ chain specific), anti-human IgG (γ chainspecific) and anti-human IgM (μ chain specific)) from Sigma ChemicalCompany (St. Louis, Mo., U.S.A.) were each diluted 1/500 in 1% BSA/PBS.One hundred μl of this mixture was added to each well. After 2 hours,wells were washed four times with PBS, then 100 μl/well of 1.0 mg ofp-nitrophenyl phosphate/ml of 1M diethanolamine-HCl (pH 9.8) containing1% BSA and 500 μM MgCl₂ was added. After 1 hour, optical densities at405 nm (O.D.) were measured using a Microplate Autoreader (Model EL 309,BIO-TEK Instruments, Winooski, Vt., U.S.A.). The O.D. results forBSA-coated wells were subtracted from the results for theglycoconjugate-BSA-coated wells for each preparation. The assays wereperformed in duplicate. The anti-human immunoglobulin conjugated toalkaline phosphatase did not bind significantly to any of the structurestested. The results are reported in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Human Antibodies Eluted from Pig Tissues Binding to Carbohydrate              Antigens                                                                      Reference                                                                             Optical Density (405 nm)                                              Number  Human Anti-Pig Kidney       Human Anti-Pig Heart                      (FIGS. 1A to 1H)                                                                      5 A.sup.o                                                                        5 B.sup.AB                                                                        6 A.sup.o                                                                        6 B.sup.AB                                                                        7 A.sup.AB                                                                        7 B.sup.o                                                                        8 A.sup.o                                                                        9 B.sup.AB                                                                        5.sup.AB                                                                         6.sup.o                                                                          7.sup.AB                                                                         9.sup.o                          __________________________________________________________________________    1       2.0                                                                              1.7 1.8                                                                              >2.0                                                                              0.8 1.8                                                                              1.2                                                                              >2.0                                                                              0.9                                                                              0.2                                                                              1.2                                                                              0.8                              2       1.9                                                                              1.3 >2.0                                                                             >2.0                                                                              0.8 1.8                                                                              1.2                                                                              >2.0                                                                              1.0                                                                              0.3                                                                              1.2                                                                              0.8                              4       1.6                                                                              1.7 >2.0                                                                             >2.0                                                                              0.6 1.6                                                                              0.9                                                                              0.6 0.8                                                                              0.2                                                                              0.8                                                                              0.7                              7       1.1                                                                              0.0 1.4                                                                              0.0 0.0 1.3                                                                              0.2                                                                              0.0 0.0                                                                              0.2                                                                              0.1                                                                              0.3                              8       1.1                                                                              0.0 1.7                                                                              0.0 0.0 1.0                                                                              0.2                                                                              0.0 0.0                                                                              0.1                                                                              0.1                                                                              1.2                              10      0.6                                                                              0.0 0.8                                                                              0.0 0.4 0.5                                                                              0.0                                                                              0.1 0.8                                                                              0.1                                                                              0.6                                                                              0.6                              11      0.2                                                                              0.1 0.3                                                                              0.0 0.1 0.0                                                                              0.0                                                                              0.0 0.1                                                                              0.1                                                                              0.3                                                                              0.8                              12      0.2                                                                              0.2 1.9                                                                              0.3 0.1 1.1                                                                              0.1                                                                              0.0 1.1                                                                              0.8                                                                              0.5                                                                              0.3                              13      0.6                                                                              0.0 0.5                                                                              0.0 0.1 0.9                                                                              0.0                                                                              0.0 0.0                                                                              0.2                                                                              0.0                                                                              0.3                              15      0.2                                                                              0.0 0.7                                                                              0.0 0.0 1.9                                                                              0.0                                                                              0.0 0.0                                                                              0.3                                                                              0.1                                                                              0.6                              16      0.1                                                                              0.0 0.6                                                                              0.0 0.1 1.6                                                                              0.1                                                                              0.0 0.1                                                                              0.3                                                                              0.1                                                                              0.5                              17      1.0                                                                              0.0 0.3                                                                              0.0 0.0 1.3                                                                              0.0                                                                              0.0 0.0                                                                              0.6                                                                              0.1                                                                              0.4                              18      0.2                                                                              0.1 1.1                                                                              0.1 0.2 0.9                                                                              0.1                                                                              0.0 0.9                                                                              0.4                                                                              0.4                                                                              0.4                              19      1.2                                                                              0.4 0.4                                                                              0.1 0.4 1.2                                                                              0.1                                                                              0.1 0.7                                                                              1.5                                                                              0.7                                                                              0.2                              20      0.8                                                                              0.4 0.7                                                                              0.4 0.3 1.9                                                                              0.1                                                                              0.0 0.6                                                                              1.6                                                                              0.8                                                                              0.4                              21      0.1                                                                              0.4 0.1                                                                              0.8 0.3 0.8                                                                              0.1                                                                              0.2 0.7                                                                              0.6                                                                              1.8                                                                              0.4                              22      0.1                                                                              0.0 1.1                                                                              0.0 0.1 0.1                                                                              0.0                                                                              0.0 0.5                                                                              0.1                                                                              0.3                                                                              0.5                              23      0.1                                                                              0.6 0.3                                                                              0.2 0.9 0.2                                                                              0.0                                                                              0.3 0.6                                                                              0.5                                                                              0.9                                                                              0.4                              24      0.2                                                                              0.0 0.2                                                                              0.0 0.7 0.1                                                                              0.0                                                                              0.1 0.2                                                                              0.3                                                                              0.4                                                                              >2.0                             25      0.5                                                                              0.7 0.4                                                                              1.0 1.1 0.5                                                                              0.1                                                                              0.1 2.0                                                                              0.4                                                                              >2.0                                                                             1.5                              26      0.4                                                                              0.3 0.2                                                                              0.4 0.9 0.1                                                                              0.0                                                                              0.1 0.5                                                                              0.2                                                                              >2.0                                                                             1.7                              27      0.1                                                                              0.1 0.2                                                                              0.2 0.1 0.6                                                                              0.0                                                                              0.1 0.3                                                                              0.1                                                                              1.1                                                                              0.5                              28      0.2                                                                              0.4 0.7                                                                              0.3 1.0 0.3                                                                              0.0                                                                              0.1 0.4                                                                              0.1                                                                              1.2                                                                              1.8                              29      0.1                                                                              0.4 0.2                                                                              0.1 0.5 0.2                                                                              0.0                                                                              0.1 1.2                                                                              0.2                                                                              0.7                                                                              0.6                              30      0.4                                                                              0.1 0.2                                                                              0.1 0.6 0.1                                                                              0.0                                                                              0.1 0.1                                                                              0.1                                                                              >2.0                                                                             0.4                              31      0.2                                                                              0.0 0.3                                                                              0.5 1.7 0.1                                                                              0.0                                                                              0.1 0.7                                                                              0.4                                                                              >2.0                                                                             0.6                              32      1.4                                                                              0.8 0.1                                                                              0.3 1.9 0.8                                                                              0.1                                                                              0.1 1.0                                                                              0.3                                                                              1.6                                                                              1.1                              __________________________________________________________________________     .sup.o Human o plasma adsorbed onto tissue                                    .sup.AB Human plasma adsorbed onto tissue                                

Numerous carbohydrate structures bound antibodies eluted from pig heart,pig kidney and/or pig red cell stroma. Some of these structures areincluded in Table 1. As shown in Table 1, B-like carbohydrate molecules(especially linear B type 2and linear B type 6) exhibited the mostreactivity for the majority of samples tested, including anti-pig heartand anti-pig kidney.

For individual eluted antibody preparations other carbohydrate antigensbound antibodies, including A or A-like carbohydrates (namely Adisaccharide, A trisaccharide, a variety of A tetrasaccharides andlinear A type 6); Forssman disaccharide and Forssman trisaccharide;α-L-Rhamnose and Rhamnose-containing structures;N-acetyl-β-D-glucosaminide (βGlcNAc) and βGlcNAc-containing structures(Table 1). However, not all of these antigens bound significant levelsof antibody for all preparations.

The populations of anti-carbohydrate antibodies varied depending on theparticular tissue and the individual serum adsorbed, but anti-linear Btype 2, anti-linear B type 6 and anti-B dissacharide antibodies werepresent in every preparation of antibodies tested (Table 1). Based onthe ELISA assay, these anti-linear B type 2, anti-linear B type 6 andanti-B dissacharide antibodies appear to be the most significant groupof anti-pig carbohydrate antibodies.

Antibodies capable of binding to A-related structures were expectedsince numerous papers have been published on many A or A-likeglycolipids and glycoproteins isolated and/or characterized from swinetissue. A and O phenotypes have been reported in pigs (Holgersson et al.1990¹²) and significant levels of anti-A are found in serum and plasmaof humans with O or B phenotypes. Some of the O.D. readings forantibodies capable of binding to synthetic A structures were lower thanexpected. For example, for human O plasma anti-pig kidney (sample 8A° inTable 1), the pig had an A-phenotype based on agglutination of pigerythrocytes with human anti-A blood typing reagent, but the O.D.readings were less than 0.2 for A trisaccharide, A type 4, A type 5 andA type 6. The O.D. readings for human O plasma anti-pig kidney (sample7B° in Table 1), however, were relatively high against A trisaccharide,A type 4, A type 5, and A type 6 (O.D.>1.0).

In most of the human anti-pig heart preparations some of the highestreadings were against N-acetyl-β-D-glucosaminide (βGlcNAc),βGlcNAc(1-4)βGlcNAc, α-L-Rhamnose (α-L-Rha) andα-L-Rha(1-3)βGlcNAc(1-2)α-L-Rha (Table 1). There may be certainpopulations of anti-carbohydrate xenoantibodies that are more importantfor some organs.

Some of the monosaccharide and oligosaccharide structures that boundsignificant levels of human antibodies eluted from pig heart, pig kidneyand/or pig red cell stroma (optical density at 405 nm≧0.5 after 1 hour)from one or more preparation are listed in Table 1.

There were variations in the carbohydrate structures which appeared tohave fairly high antibody binding (O.D. at 405 nm≧0.5) from preparationto preparation. The type of human plasma, the individual plasma, theindividual animal and/or the organ from which the antibodies were elutedall appeared to create variation.

The findings set forth in this application will apply to other organsand cells that have not been investigated. One specific embodimentcontemplated by this invention is its use in conjunction with pancreaticislet cell xenotransplantation.

EXAMPLE 2 Measuring Human Hemolytic Antibodies to Porcine, Bovine andSheep Erythrocyte Antigens

Heat-Inactivation of serum and plasma for Examples 2, 3 and 4

Heat inactivated human serum or human plasma was used unless otherwisenoted. It was heated in a water bath at 56° C. for 30 minutes toinactivate the complement (heat-inactivated).

Preparation of Serum for Example 2

Portions (1.2 ml) of human Type O serum were then transferred into testtubes containing 0.4 g±0.01 g of Chromosorb P (Manville Corp., Denver,Colo.), synthetic carbohydrate structures bound to Chromosorb P(hereinafter called SYNSORB, trademark of Chembiomed Ltd.), or mixturesof carbohydrate structures bound to Chromosorb P (mixtures of SYNSORB);and 0.3 ml PBS. Example 6, infra, describes the technique for couplingthe carbohydrates to Chromasorb P as applied to compound 2 of FIG. 1A.The samples were mixed and incubated at 4° C. for 16 hours. Thesepreparations were centrifuged at 1000 rpm and the supernatantscollected.

Preparation of erythrocytes

Pig, bovine and sheep erythrocytes were washed 3 times with excess PBS(using a 1 minute centrifugation at 1000×g after each wash), then washed3 times with 1% BSA in PBS. The final cell pellet was diluted to 1%(v/v) in 1% BSA in PBS.

Hemolytic assay

A hemolytic assay was then performed based on an assay described by Rappand Borsos (1966)²¹. The adsorbed and unadsorbed serum samples werediluted 1/4 in 1% BSA in PBS, then 50 μl was added to the appropriatewells (FIG. 2 results); or adsorbed and unadsorbed human serum sampleswere undiluted or diluted 1/2 or 1/4 with 1% BSA in PBS, then 100 μladded to the appropriate wells (FIG. 3 results). A 1% suspension ofporcine erythrocytes (FIG. 2) or a 2% suspension of porcine, sheep orbovine erythrocytes (FIG. 3) was added to the appropriate wells of a Vshaped 96 well microtitration plate(s). Several control wells were alsoprepared containing 50 μl of erythrocyte suspension and 50 μl or 100 μlof 1% BSA in PBS. After a 1 hour incubation at 22° C. the plate(s) werecentrifuged for 5 minutes at 1100 rpm in a Beckman TJ-6 Centrifuge(Beckman Instruments, Inc., Palo Alto, Calif., U.S.A.). A complementpreparation was prepared using 1 ml of lyophilized LOWTOX-M rabbitcomplement (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada)reconstituted with 1 ml of cold, purified water, then diluted 1/15 withgelatin veronal buffer (Sigma Chemical Co.). The supernatants wereremoved, then 200 μl of diluted complement, gelatin veronal buffer, orwater was added to the wells. The samples were mixed using a cleanpipette tip for each well, then incubated for 1 hour at 37° C. Theplate(s) was centrifuged at 2000 rpm for 3 minutes, then 150 μl ofsupernatant/well was transferred into a flat-bottomed plate and theoptical density at 405 nm (O.D.) was read using a Microplate Autoreader(Model EL 309, BIO-TEK Instruments, Winooski, Vt., U.S.A.).

The percentage (%) hemolysis was calculated based on: 0% hemolysis=theaverage O.D. from wells to which erythrocytes, 1% BSA in PBS andcomplement were added (no serum); and 100% lysis=the average O.D. fromwells to which erythrocytes, unadsorbed serum or serum absorbed withChromosorb P and complement were added.

Adsorption of human serum with particular carbohydrate structures boundto matrix and combinations of matrix-bound carbohydrates reduced thehemolysis of porcine, sheep and bovine erythrocytes (FIGS. 2 and 3).

The samples in FIG. 2 were adsorbed with the following SYNSORB-boundcarbohydrates: a combination of linear B type 6, A trisaccharide, Btrisaccharide and βGlcNAc (1); a combination of linear B type 6 andβGlcNAc (2); a combination of linear B type 6 and B trisaccharide (3); acombination of linear B type 6 and A trisaccharide (4); βGlcNAc (5);linear B type 2 linear B type 6 (7); B trisaccharide (8); Atrisaccharide (9); LacNAc (10); and Chromosorb P alone (11). Structuresare shown in FIG. 1A to 1H.

The samples in FIG. 3 were adsorbed with the following SYNSORB-boundcarbohydrates: a combination of linear B type 6, A trisaccharide, Btrisaccharide and βGlcNAc (1); a combination of linear B type 6 and Btrisaccharide (2); a combination of linear B type 6 and βGlcNAc (3); acombination of linear B type 6 and A trisaccharide (4); linear B type 2(5); βGlcNAc (6); linear B type 6 (7); B trisaccharide (8); Atrisaccharide (9); LacNAc (10); and Chromosorb P alone (11).

As shown in FIG. 3, the combination of A trisaccharide-SYNSORB andlinear B type 6-SYNSORB were more effective at reducing hemolyticactivity to porcine and sheep erythrocytes than either linear B type6-SYNSORB or A trisaccharide separately. Not all sera have anti-Aactivity and there is some diversity in the specificity andconcentration of antibodies from individuals with various blood typesand between individuals with the same blood type. Therefore it may benecessary to employ a combination of monosaccharides and/oroligosaccharides to reduce the majority of cytolytic antibodies. Thiswould be the best combination of carbohydrate structures from person toperson, from species to species, and from organ to organ.

EXAMPLE 3 Removal of Human Cytotoxic Antibodies to Pig Antigens withMatrix-Bound Carbohydrates

In this experiment, pig erythrocytes, lymphocytes and an adherent pigkidney cell line (LLC-PK₁ from American Type Culture Collection,Rockville, Md., U.S.A.: Cat.#CRL 1392-CL101) were used as target cells.The erythrocytes and lymphocytes were separated from whole heparinizedpig blood with Ficoll-Paque (Pharmacia LKB Biotechnology Inc.,Piscataway, N.J., U.S.A.: Cat.#17-0840-02). The pig kidney cell line wascultured in Medium 199 (Gibco BRL Canada, Burlington, Ontario, Canada)with 10% FBS (Hybri-Max from Sigma Chemical Co.: Cat.#CPSR-3). Each ofthese target cells was labelled with chromium-51 (Amersham, Oakville,Ontario, Canada: Cat.#CJS.11) by incubation at 37° C. for 1 hour (10⁶cells with 100 μl (100 μCi) of chromium). Cells were washed once inmedium, incubated for 1 hour at 37° C., and then washed twice to removenon-incorporated chromium.

Undiluted heat-inactivated plasma (100 μl) was incubated with 100 μl ofchromium-labelled target cells (2×10⁴ cells) in 96 well V bottommicrotiter plates (Dynatech Lab., Chantilly, Va., U.S.A.:Cat.#1-220-25X) at 37° C. for 1 hour with shaking (20 rpm). A 1/15dilution of rabbit complement (LOWTOX-M from Cedarlane LaboratoriesLtd., Hornby, Ontario, Canada: Cat.#(L3051) was added to each well. Theplate was again incubated for 1 hour at 37° C. with shaking. Plates werethen spun at 1000 rpm and supernatants were counted for chromium contentin the Beckman gamma 4000 counter (Beckman Instruments, Inc., Fullerton,Calif., U.S.A.). Spontaneous and total release controls were also run.Spontaneous counts were always less than 5% of the total counts, andtypical total counts of cells ranged from 10,000 to 20,000 cpm.

As seen from the results shown in FIGS. 4A and 4B, unadsorbed human Oand AB plasma contained antibodies capable of lysing chromium-labelledpig lymphocytes, erythrocytes, and the pig kidney cell line. Perfusionof these plasma samples through the heart reduced cytotoxic activity(FIGS. 4A and 4B) presumably due to the binding of cytotoxic antibodiesto the pig tissues. In a transplant situation, the binding of theseantibodies could potentially cause hyperacute rejection.

To investigate the ability of the carbohydrates (A trisaccharide, Btrisaccharide, LacNAc (βGal(1-4) βGlcNAc-R), linear B type 2 and linearB type 6), coupled onto SYNSORB, to remove human anti-pig cytotoxicantibodies, 1 ml of plasma was adsorbed overnight at 4° C. with 0.2 g ofthe different SYNSORB. The adsorbed plasma was then tested in thechromium release assay. The A trisaccharide-SYNSORB, Btrisaccharide-SYNSORB, LacNAc-SYNSORB and Chromosorb P had little effectin these examples (FIGS. 4A and 4B). Linear B type 2-SYNSORB and linearB type 6-SYNSORB significantly reduced the cytolytic activity. Theseresults clearly demonstrate that linear B type 2 or linear B type 6bound to SYNSORB is effective in binding human anti-pig cytotoxicantibodies.

The following experiments were conducted using the experimentalprocedures described above, except that the plasma used were not heatinactivated. Rabbit complement was added to each well, as describedpreviously.

Human plasma (A, B, O and AB) samples were adsorbed with matrix-boundcarbohydrates (SYNSORBS) to remove human anti-pig cytotoxic antibodies.(Refer to FIGS. 1A to 1H for structures of the carbohydrate compoundsused.) The adsorbed plasma were tested in the chromium release assay.These results, shown in FIG. 5, clearly demonstrate that the Bdisaccharide (formula 4 from FIG. 1A) and Linear B type 6 (formula 2from FIG. 1A) SYNSORBS significantly reduce the cytotoxic activity inall human blood groups.

Four different AB human plasma types were adsorbed with matrix-boundcarbohydrates (SYNSORBS) to remove human anti-pig cytotoxic antibodies.(Refer to FIG. 1A to 1H for structures of the carbohydrate compoundsused.) The adsorbed plasma were tested in the chromium release assay.Results are shown in FIG. 6. The B disaccharide and Linear B type 6SYNSORBS significantly reduced the cytotoxic activity in one particularAB plasma (1808035). In another plasma sample (1901422), these SYNSORBSshowed only marginal reduction of cytotoxic activity. In sample 1809451,the linear B type 6 SYNSORB reduced cytotoxic activity, but the Bdisaccharide SYNSORB did not. In sample 7708213, neither the Bdisaccharide SYNSORB nor the linear B type 6 SYNSORB significantlydecreased cytotoxic activity. This example demonstrates heterogeneity inthe population, since the cytotoxic antibodies found in some plasmaapparently require adsorption by the αGal(1-3)βGal(1-4)βGlc structurewhile the Linear B backbone of the B disaccharide (αGal(1-3)βGal) issufficient for adsorption of the cytotoxic antibodies in other plasma.

Human plasma (A, B, O, and AB) samples were adsorbed with matrix-boundcarbohydrates (SYNSORBS) to remove human anti-pig cytotoxic antibodies.(Refer to FIGS. 1A to 1H for structures of the carbohydrate compoundsused.) The adsorbed plasma were tested in the chromium release assay.The results are shown in FIG. 7. Linear B type 6 and Linear B type 1(formula 3 from FIG. 1A) SYNSORBS significantly reduced the cytotoxicactivity in human plasma samples. P-K trisaccharide (formula 37 fromFIG. 1H) SYNSORB marginally reduced cytotoxic activity in the Type ABand B human plasma tested. P-1 trisaccharide (formula 40 from FIG. 1H)SYNSORB marginally reduced cytotoxic activity in this Type A humanplasma. P-K and P-1 trisaccharide SYNSORBS did not show reduction ofcytotoxic activity in this Type O human plasma. These results show thatthe Linear B structure is specific for cytotoxic antibodies, but othercarbohydrates may remove or block other antibody specificities.

EXAMPLE 4 Removal of Human Cytotoxic Antibodies to Pig Antigens withCarbohydrate Haptens

This procedure was done to investigate the potential for inhibitinghuman preformed cytotoxic antibodies with carbohydrate(s) haptens. Humanheat inactivated plasma (1 ml) was incubated overnight at 4° C. with0.215 g of A-trisaccharide-X-Y, B-trisaccharide-X-Y, βGlcNAc-X-Y,LacNAc-X-Y, linear B type 2-X-Y, or linear B type 6-X-Y (X-Y═--O--(CH₂)₈--COOCH₃ or --O--(CH₂)₈ --COOCH₂ CH₃, as in FIGS. 1A to 1H, referred toas SYNJECT, trademark of Chembiomed, Ltd.). The chromium release assaywas then performed with treated and untreated plasma; the pig kidneycell line (LLC-PK₁) was used as the target cell. As depicted in FIGS. 8Aand 8B, linear B type 2-X-Y and linear B type 6-X-Y completely inhibitedthe cytotoxic effect of antibodies to the pig cell line. Other haptenspartially inhibited these cytotoxic antibodies (i.e., B trisaccharide).These results indicate that a soluble carbohydrate(s) hapten (SYNJECTform) may be effective for in situ inhibition of pre-formed humananti-carbohydrate antibodies.

The following experiments were conducted using the experimentalprocedures described above, except that the plasma used were not heatinactivated. Rabbit complement was added to each well, as describedabove in Example 3.

Human plasma (A, B, O, and AB) samples were incubated with carbohydratehaptens to inactivate human anti-pig cytotoxic antibodies. (Refer toFIGS. 1A to 1H for structures of the carbohydrate compounds used.) Theincubated plasma were tested in the chromium release assay. Results areshown in FIG. 9. B disaccharide and Linear B type 6 haptenssignificantly reduced the cytotoxic activity in all blood groups.

Two different AB human plasma types were incubated with carbohydratehaptens to remove human anti-pig cytotoxic antibodies. (Refer to FIGS.1A to 1H for structures of the carbohydrate compounds used.) Theincubated plasma were tested in the chromium release assay. The resultsare shown in FIG. 10. B disaccharide hapten only marginally reduced thecytotoxic activity in both plasma types. Linear B type 6 and B type 6haptens each reduced the cytotoxic activity in one plasma type, but notthe other. This example further demonstrates heterogeneity in thepopulation.

EXAMPLE 5 Preparing (αGal(1-3)βGal(1-4)βGlc-O-(CH₂)₈ CONH)₁₇ -BSA

Sixty milligrams of the methyl ester form of compound 2 in FIG. 1(αGal(1-3)βGal(1-4)βGlc-O-(CH₂)₈ COOCH₃) was dissolved in 1.5 ml ofhydrazine hydrate. After 2 hours, the hydrazine hydrate was removed inhigh vacuum and the residue was co-evaporated with water, then thehydride was purified on a C18 reverse phase column (WatersChromatography Division, Millipore Corp., Milford Mass., U.S.A.) thenfreeze-dried. Nineteen mg of the synthetic hapten hydride was dissolvedin 0.2 ml dry amine-free DMF and cooled to -20° C. under an inertatmosphere. A 3.6M solution (31 μl) of HCl in dioxane and 5.3 μl (45μmoles) of tertiary butyl nitrite were added and the solution stirredfor 30 minutes at 20° C. Sulfamic acid (1.4 mg) in DMF was added andstirred for an additional 15 minutes. This acyl azide solution was addeddirectly to a solution of (30 mg BSA in 3 ml 0.2M N-thioldiethanolaminein water, pH 8.98, 0° to 4° C.). After 16 hours at 4° C., the solutionwas dialyzed against water, then freeze dried. Thirty-six mg of a whitepowder was recovered. The incorporation of hapten was determined by thephenol-sulfuric acid method (Dubois et al. 1956⁵) and was calculated tobe 17 moles of hapten per mole of BSA.

This preparation was used in Example 1 to detect anti-linear B type 6antibodies.

EXAMPLE 6 Preparation of an Effective Immunoadsorbent(αGal(1-3)βGal(1-4)βGlc--O--(CH₂)₈ CONH)-SYNSORB

Forty mg of the hydride form of compound 2, in FIG. 1A was prepared asdescribed in Example 5 and dissolved in 0.3 ml DMF. This solution wascooled to -20° C. under an inert atmosphere. A 3.6M solution (62 μl) ofHCl in dioxane and 11 μl (90 μmoles) of tertiary butyl nitrite wereadded and the solution stirred for 30 minutes. Hunigs base(N,N-Diisopropylethylamine from Aldrich Chemical Co., Inc., Milwaukee,Wis. U.S.A.: Cat.#D12.580.6) (45 μl or 258 μmoles) was added and stirredfor an additional 2 minutes. This acyl azide solution was added directlyto a slurry of silylaminated calcined diatomaceous earth (56 g ofChromosorb P from Manville Corp., Denver, Colo.) in acetonitrile (150ml) at 4° C. (The silylamination was performed using a protocoldescribed by Weetall (1976)²⁴.) After stirring for 2 hours at 4° C. and2 hours at room temperature, the solid was collected by filtration andthe unreacted amines were N-acetylated using 5% acetic anhydride inmethanol, gently stirred for one hour, then incubated overnight at roomtemperature. The solid portion was then washed using methanol and ethylether, then air dried. The incorporation of hapten was determined by thephenol-sulfuric acid method and determined to be 0.87 μmole of hapten/gof matrix.

This preparation was used in Examples 2 and 3 to remove anti-linear Btype 6 antibodies.

EXAMPLE 7 Detection of a Reduction of Human Antibody Titers toCarbohydrate Compounds by Plasma Adsorption with Matrix-BoundCarbohydrate(s) Using Enzyme-Linked Immunosorbent Assay (ELISA)

Flat-bottom 96 well immuno-plates (Gibco-BRL, Burlington, Ontario,Canada) were coated with 100 μl/well of 10 μg/ml BSA or carbohydrate-BSAconjugates diluted in 0.05M carbonate-bicarbonate coating buffer (SigmaChemical Co.), pH 9.6. Plates were incubated at 4° C. for 18 hours, thenemptied and blocked with 150 μl/well of 1% BSA (Sigma) in PBS for onehour at room temperature. The plates were then washed three times withPBS containing 0.05% polyoxyethylene-sorbitan monolaurate (tween 20;Sigma).

Human plasma from blood types A, B, AB, and O, either unadsorbed oradsorbed with Chromosorb P, B dissacharide SYNSORB, or Linear B type 6SYNSORB were diluted 1:100 with PBS-tween 20 and aliquoted in triplicateto BSA control on carbohydrate-BSA coated wells. After an incubationperiod of two hours at room temperature and three subsequent washingswith PBS-tween 20, all wells received 100 μl of goat anti-humanpolyvalent (α, γ, and μ chain specific)-alkaline phosphate conjugate(Sigma) diluted 1:350 in PBS-tween 20. Once again the plates wereincubated at room temperature for two hours and then washed three timeswith PBS-tween 20. One hundred microliters of disodium p-nitrophenylphosphate (Sigma) diluted to 1 mg/ml in 10% (v/v) diethanolamine buffer,pH 9.8 (Fisher Scientific), containing 0.01% (w/v) magnesium chloride(Fisher Scientific) was added to each well. The immuno-plates wereincubated in the dark at room temperature for 30 minutes. The absorbanceat 405 nm was then read for each well using a Titertek Multiskan (FlowLaboratories, McLean, Va.). Measurements of non-specific binding ofplasma to wells coated with BSA only, were subtracted from the resultspresented in FIGS. 11A-11D and 12A-12D.

Human A, B, O, and AB plasma adsorbed with SYNSORBS were tested againsta panel of Hapten-BSA conjugates. Refer to FIGS. 1A and 1H forstructures of the carbohydrate compounds used. As shown in FIG. 11A-11D,plasma adsorbed with B dissacharide or Linear B type 6 SYNSORBS showedmarkedly decreased binding to the B dissacharide, Linear B type 6 andLinear B type 2 BSA conjugates when compared to unadsorbed plasma orplasma adsorbed with Chromosorb P. These SYNSORBS did not decreasebinding to N-acetyl-β-D-Glucosaminide (formula 25 from FIG. 1F) andα-L-Rhamnose (formula 31 from FIG. 1H) BSA conjugates. These resultsdemonstrate that B dissacharide and Linear B type 6 SYNSORBS were ableto remove or reduce specific antibody from plasma.

Human AB plasma adsorbed with SYNSORBS were tested against a panel ofHapten-BSA conjugates. Refer to FIGS. 1A to 1H for structures of thecarbohydrate compounds used. As shown in FIG. 12A-12D, plasma adsorbedwith B dissacharide or Linear B type 6 SYNSORBS showed decreased bindingto the B dissacharide, Linear B type 6 and Linear B type 2 BSAconjugates when compared to unadsorbed plasma or plasma adsorbed withChromosorb P. These SYNSORBS did not decrease binding to otherHapten-BSA conjugates. These results demonstrate that B dissacharide andLinear B type 6 SYNSORBS were able to remove or reduce specific antibodyfrom plasma.

Modifications of the above-described modes that are obvious to those ofskill in the fields of transplantation immunology, carbohydratechemistry, and related fields are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. An immunoadsorbent composition useful forremoving xenoantibodies from the blood of a human recipient of axenograft to attenuate the rejection of the xenograft by the recipientcomprising a biocompatible solid support having at least one identifiedcarbohydrate xenoantigen comprising linear B type 6 to preformedantibodies attached thereto through a biocompatible linker arm.
 2. Thecomposition of claim 1 wherein the carbohydrate xenoantigen furthercomprises at least one of the glycosides having structures 3, 21, 23,26, 27, 28, 29, 30, 31 and 32 of FIGS. 1A through 1H where X is selectedfrom the group consisting of O, S, NH, and a bond and Y is an aglycongroup.
 3. The composition of claim 2 wherein Y represents the groupconsisting of --(A)--Z wherein A represents a bond, an alkylene group offrom 2 to 10 carbons or --(CH₂ --CR₁ --G)_(n) --, wherein n is aninteger equal to 1 to 5 inclusive; R₁ is selected from the groupconsisting of hydrogen, methyl and ethyl; and G is selected from thegroup consisting of hydrogen, oxygen, sulfur, nitrogen, phenyl, andphenyl substituted with 1 to 3 substituents selected from the groupconsisting of amine, hydroxyl, halo, and alkyl of from 1 to 4 carbonatoms, and Z is selected from the group consisting of hydrogen, methyland when G is not oxygen, sulfur or nitrogen and A is not a bond, then Zis also selected from the group consisting of OH, SH, --NH₂, --NHR₂,--C(O)OR₂, --C(O)NH₂, --C(O)NH--NHR₂, --C(O)NHR₂ and --C(O)N(R₂)₂wherein each R₂ is independently hydrogen or alkyl of from 1 to 4 carbonatoms.
 4. The composition of claim 2 wherein X is --O-- and Y is --A--Zwhere A is alkylene of 2 to 10 carbon atoms and Z is selected from thegroup consisting of --C(O)OR₂, --C(O)NH₂ and --C(O)NHR₂ where R₂ ishydrogen or alkyl of from 1 to 4 carbon atoms.
 5. The composition ofclaim 1 wherein the carbohydrate xenoantigen further comprises theglycoside structure 1 of FIG. 1A and X is --O-- and Y is --A--Z where Ais alkylene of 2 to 10 carbon atoms and Z is selected from the groupconsisting of --C(O)OR₂, --C(O)NH₂ and --C(O)NHR₂ where R₂ is hydrogenor alkyl of from 1 to 4 carbon atoms.
 6. The composition of claim 1wherein the carbohydrate xenoantigen comprises the glycoside structure 2of FIG. 1A and X is --O-- and Y is --A--Z where A is alkylene of 2 to 10carbon atoms and Z is selected from the group consisting of --C(O)OR₂,--C(O)NH₂ and --C(O)NHR₂ where R₂ is hydrogen or alkyl of from 1 to 4carbon atoms.
 7. The composition of claim 1 wherein the carbohydratexenoantigen further comprises the glycoside structure 4 of FIG. 1A and Xis --O-- and Y is --A--Z where A is alkylene of 2 to 10 carbon atoms andZ is selected from the group consisting of --C(O)OR₂, --C(O)NH₂ and--C(O)NHR₂ where R₂ is hydrogen or alkyl of from 1 to 4 carbon atoms.